1. Field of the Invention
The present invention is directed to an optical microscope having an adjustable optical condenser annulus and, more particularly, to a phase contrast or inverted microscope using an electrical device to construct a dynamically adjustable visible condenser annulus for viewing transparent living cells under a liquid.
2. Description of Related Art
The present invention relates to a phase contrast microscope for viewing transparent living cells under a liquid. In this configuration of the microscope, called an inverted microscope, the samples of cells are placed in a micro plate under a liquid. Living cells are placed on a plate on the microscope and a light from above the well illuminates the sample. The cells are typically at the bottom of the well. The micro plate well has a clear bottom and the objective, located below the well, is focused on the cells. The focused light then travels down into the microscope to the eyepiece or off to a camera for viewing the cells for discovery.
Live cells are transparent and difficult to see without a means to enhance the image. A method called Phase Contrast Imaging is common technique for improving the contrast of the cell. The visible light used to illuminate the cells is first passed through a condenser annulus, which is a piece of metal with a stamped circular ring, where light can pass to produce the bright or visible phase ring. This ring is specific to the objective used for magnifying the cell for observation. After the visible condenser annulus, the bright phase ring travels through a condenser lens to collimate the light and then on to the sample plane. If there is no sample in the plane, the light travels on through the objective where the light is magnified. In a phase contrast objective, below the magnification lens, is a black ring. If there is no distortion to the light, this dark phase ring blocks the light from the bright condenser annulus.
Alignment of the bright or light phase ring from the condenser annulus, with the dark phase ring is necessary for getting good phase when viewing transparent cells under the microscope. The alignment method involves switching to a Bertrand lens in the eyepiece. The researcher switches a lens and ring into the eyepiece and manually adjusts, usually via adjustment knobs, a joystick, a computer, and the like to adjust the visible condenser annulus to align the visible ring with the black ring. With the sample in the light path, the transparent cell causes the light ring to be refracted, resulting in misalignment with the dark phase ring. This misalignment causes constructive interference, producing a bright area. There is typically greater refraction at the edges of cells, producing a bright edge and improving the contrast in the image. Cells that are typically very difficult to see under the microscope can be observed and imaged to a much finer detail by using the Phase Contrast Imaging method.
In the case where one is observing live cells under a liquid, the light is distorted by the liquid before reaching the cell and phase imaging is greatly compromised. Since the meniscus of the liquid is typically well defined, the meniscus acts like a lens aligned with the objective only in the middle of the sample well. In the middle of the well, a reduced but useable version of phase imaging is possible, however, care must be taken to ensure that the image is taken from the middle of the well, otherwise, the image is not useable.
Micro plates come in many different configurations having 6, 12, 24, 48, 96, 384, and 1536 wells. In a 6-well plate, a single well is 1.4″ (3.56 cm) in diameter. The meniscus is more pronounced at the edge and the liquid flattens out toward the center of the well. Phase imaging of a live cell under the liquid at the center of a 6-well plate is good and is used by many researchers for their live cell studies. However, during the course of observation of the live cells, the distribution of the cells may change causing one to move away from the center of the well. Accordingly, the phase will start to change or reduce. The further away one is, the less detail that can be seen in the cells. Also, if one were using a micro plate having smaller wells, such as a 96-well plate, for example, the well diameter is 0.26″ (0.66 cm). In this case, the rneniscus lens effect is pronounced. Thus, one can only get minimal phase at the very center of the well. For micro plates with higher number wells, which are significantly smaller, such as micro plates a number wells of 384 and 1536, there is no phase. Hence, use of these micro plates having a higher number of wells would be difficult, if not impossible.
In some cases, it is possible to manually adjust the phase ring while observing cells in a well, even off-center, and get some phase to help with viewing the cells. This method is obviously very helpful, but this changes with location and changes from well to well during a series of observations. The fluid height and viscosity of the liquid in the well also change during the course of an observation, which can take several days or span a series of wells in a micro plate, and can change the effect when viewing with phase. A dynamic means is required to compensate for these changes in the light path when viewing live cells under a liquid over the course of the observation or over a series of observations.
U.S. Pat. No. 5,751,475 to Ishiwata, which is incorporated by reference in its entirety, discloses a phase contrast microscope that uses an Liquid Crystal Display (LCD) in place of the dark phase ring. The goal of Ishiwata is to provide both phase imaging and standard visible light imaging without changing the phase ring. The reference discusses two modes of operation of the microscope, with phase imaging and without phase imaging for specimen inspection. The background portion of Ishiwata refers to Japanese Patent Application No. 27-2523 as being directed to a proposed method of adjusting a contrast of a phase contrast image by changing a phase difference and transmissivity at a phase ring by using a polarizing plate which is rotated. However, the rotation of this polarizing plate requires a separate mechanical member.
U.S. Pat. No. 5,969,853 to Takaoka, which is incorporated by reference in its entirety, discloses an optical microscope having optical modulation elements to produce various changes to the dark ring for contrast enhancement by providing regions for high and low transmittance.
There is a need in the art for an improved phase contrast imaging system for use in inverted microscopes over a wide portion of a micro plate when viewing through a liquid. There is also a need in the art for a system which allows for turning the visible light for imaging on and off; a system or device for providing a variety of wavelengths for visible imaging; and a system for dynamically adjusting the visible condenser annulus based upon the well size and number of wells of the particular micro plate being used and also based upon the position, fluid height and/or viscosity of the sample.